Vincent J. Cristofalo

Replicative senescence: a critical review

Human cells in culture have a limited proliferative capacity. After a period of vigorous proliferation, the rate of cell division declines and a number of changes occur in the cells including increases in size, in secondary lysosomes and residual bodies, nuclear changes and a number of changes in gene expression which provide biomarkers for senescence. Although human cells in culture have been used for over 40 years as models for understanding the cellular basis of aging, the relationship of replicative senescence to aging of the organism is still not clear. In this review, we discuss replicative senescence in the light of current information on signal transduction and mitogenesis, cell stress, apoptosis, telomere changes and finally we discuss replicative senescence as a model of aging in vivo.

Loss of HuR Is Linked to Reduced Expression of Proliferative Genes during Replicative Senescence

ABSTRACT Cellular aging is accompanied by alterations in gene expression patterns. Here, using two models of replicative senescence, we describe the influence of the RNA-binding protein HuR in regulating the expression of several genes whose expression decreases during senescence. We demonstrate that HuR levels, HuR binding to target mRNAs encoding proliferative genes, and the half-lives of such mRNAs are lower in senescent cells. Importantly, overexpression of HuR in senescent cells restored a “younger” phenotype, while a reduction in HuR expression accentuated the senescent phenotype. Our studies highlight a critical role for HuR during the process of replicative senescence.

Cellular replicative capacity correlates primarily with species body mass not longevity

Although the limited replicative capacity of human fibroblasts in culture is frequently used as a model for aging, a question of major interest is whether the relationship between in vitro fibroblast proliferative capacity and species longevity is primary or secondary to a relationship with species body size. In this report we establish that body mass is the primary correlative of proliferative potential rather than species life-span.

Modulation of Replicative Senescence of Diploid Human Cells by Nuclear ERK Signaling

Normal somatic cells have a limited replicative lifespan, and serial subcultivation ultimately results in senescence. Senescent cells are irreversibly growth-arrested and show impaired responses to mitogens. Activation of the ERK signaling pathway, an absolute requirement for cell proliferation, results in nuclear relocalization of active ERKs, an event impaired in senescent fibroblasts. This impairment coincides with increased activity of the nuclear ERK phosphatase MKP2. Here we show that replicative lifespan can be altered by changes in nuclear ERK activity. Ectopic expression of MKP2 results in premature senescence. In contrast, knock-down of MKP2 expression, through transduction of MKP2 sequence-specific short hairpin RNA, or expression of the phosphatase resistant ERK2(D319N) mutant, abrogates the effects of increased endogenous MKP2 levels and senescence is postponed. Nuclear targeting of ERK2(D319N) significantly augments its effects and the transduced cultures show higher than 60% increase in replicative lifespan compared with cultures transduced with wt ERK2. Long-lived cultures senesce with altered molecular characteristics and retain the ability to express c-fos, and Rb is maintained in its inactive form. Our results support that MKP2-mediated inactivation of nuclear ERK2 represents a key event in the establishment of replicative senescence. Although it is evident that senescence can be imposed through multiple mechanisms, restoration of nuclear ERK activity can bypass a critical senescence checkpoint and, thus, extend replicative lifespan.

Expression of hydrogen peroxide and glutathione metabolizing enzymes in human skin fibroblasts derived from donors of different ages

We have examined the activities and mRNA abundance of two hydrogen peroxide metabolizing enzymes (glutathione peroxidase and catalase), glutathione concentration, and the activities of several enzymes that influence glutathione concentration, including glutathione reductase (GR), glucose‐6‐phosphate dehydrogenase (G‐6‐PD), and γ‐glutamylcysteine synthetase (γ‐GCS), in 29 skin fibroblast lines derived from donors ranging in age from 14 gestational weeks to 94 years of age. H2O2 metabolizing enzyme activities and mRNA abundances were greater in skin fibroblast cultures established from postnatal donors than in fetally derived cultures. There were no significant differences in either of these parameters in cell lines established from postnatal donors of different ages. Total glutathione concentration decreased with age, but GR activity appeared to be unaffected by age. In order to estimate the ability of the cultures to produce NADPH (an important component of cellular redox status and a cofactor for GR), we determined glucose‐6‐phosphate dehydrogenase activity and mRNA abundance. We were unable to directly measure γ‐GCS activity or mRNA abundance in any of the skin lines or in fetal lung fibroblasts; however, we were able to indirectly demonstrate the presence of this enzyme by stimulating fetal lung fibroblasts with H2O2 following treatment with L‐buthionine‐S,R‐sulfoximine (BSO), an inhibitor of γ‐GCS activity. These results show that some, but not all, age‐associated differences in antioxidant defense levels are maintained in a culture environment and are consistent with the hypothesis that developmental stages of life are associated with lower antioxidant defense levels than are present in postnatal phases of life.

Differential gene expression between young and senescent, quiescent WI-38 cells

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.

Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts.

Cellular senescence is characterized by impaired cell proliferation. We have previously shown that, relative to the young counterpart, senescent WI-38 human fibroblasts display a decreased abundance of active phosphorylated ERK (p-ERK) in the nucleus. We have tested the hypothesis that this is due to elevated levels of nuclear MAP kinase phosphatase (MKP) activity in senescent cells. Our results indicate that the activity and abundance of MKP-2 is increased in senescent fibroblasts, compared to their young counterparts. Further analysis indicates that it is MKP-2 protein, but not MKP-2 mRNA level, that is increased in senescent cells. This increase is the result of the increased stability of MKP-2 protein against proteolytic degradation. The degradation of MKPs was impaired by proteasome inhibitors both in young and old WI-38 cells, indicating that proteasome activity is involved in the degradation of MKPs. Finally, our results indicate that proteasome activity, in general, is diminished in senescent fibroblasts. Taken together, these data indicate that the increased level and activity of MKP-2 in senescent WI-38 cells are the consequence of impaired proteosomal degradation, and this increase is likely to play a significant role in the decreased levels of p-ERK in the nucleus of senescent cells.

Role of the Raf/MEK/ERK and the PI3K/Akt(PKB) pathways in fibroblast senescence

Replicative senescence is characterized by numerous phenotypic alterations including loss of proliferative capacity and numerous changes in gene expression such as impaired serum inducibility of the immediate early gene c-fos and increased expression of collagenase. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors serum response factor (SRF) and ternary complex factor (TCF). TCF is activated after phosphorylation by the Extracellular signals Regulated Kinase 1 and 2 (ERK1/2), two kinases of the Raf/MEK/ERK signaling pathway. We have previously demonstrated that collagenase expression is under positive regulation by the transcription factor FKHRL1 and that this transcription factor is under negative regulation by the phosphatidylinositol 3-kinase(PI3K)/Akt(PKB) pathway. Although total activity of ERK and Akt was similar in total cell lysates from early and late passage fibroblasts our data indicate that in senescent cells neither ERK nor Akt are able to phosphorylate efficiently their nuclear targets. Our findings suggest that although they can be fully activated in the cytosol of both early and late passage cells, the Raf/MEK/ERK and the PI3K/Akt pathways, which are essential for cellular proliferation, are down regulated in the nuclei of senescent cells.

Putative role for EPC‐1/PEDF in the G0 growth arrest of human diploid fibroblasts

EPC‐1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast‐like (HDF) cells and is diminished with proliferative senescence in vitro. EPC‐1 expression in HDF cells is induced under conditions of density‐dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC‐1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC‐1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC‐1 antibodies alone enhance the ability of near plateau‐phase early PDL WI‐38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC‐1 (rEPC‐1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC‐1 expression in WI‐38 and IMR‐90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC‐1 expression with SV40 transformation occurs at the level of steady‐state mRNA and protein accumulation with genomic EPC‐1 sequences grossly intact. Taken together, these results suggest that EPC‐1 may play a role in the entry of early passage fibroblasts into a G0 state or the maintenance of such a state once reached.

Significant correlation of species longevity with DNA double strand break recognition but not with telomere length.

The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSBs) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed.